Chemoreceptors expressed in taste, olfactory and male reproductive tissues

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Gene 178 (1996) 1 5

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Chemoreceptors expressed in taste, olfactory and male reproductive tissues 1 Mary Beth Thomas *, Susan L. Haines 2, Richard A. Akeson Children's Hospital Research Foundation, 3333 Burner Avenue, Developmental Biology Department, Cincinnati, OH 45229 USA Received 1 November 1995; revised 26 January 1996; accepted 30 January 1996

Abstract

We have identified three genes encoding previously uncharacterized chemoreceptors expressed in rat sensory and reproductive tissues using a reverse transcriptase polymerase chain reaction strategy. Degenerate oligonucleotides designed from conserved sequences in the rat olfactory receptor gene family were used to amplify candidate receptor gene products expressed in taste tissue. Sequence analysis of three distinct clonal isolates revealed that the gene products from taste bud were 30-75% identical to previously identified olfactory receptor genes. The genomic coding sequences predicted protein structures with seven membrane spanning regions that have strong conservation relative to other members of the G-protein-coupled olfactory receptor gene family. Transcripts for each of the three gene products were detected exclusively in taste, olfactory and male reproductive tissue. Sequence analysis of the polymerase chain reaction products confirmed that identical transcripts were expressed in all three tissues. These findings are the first demonstration that identical olfactory receptor-like gene are expressed in three distinct tissues. Keywords: Seven transrnembrane domain receptors; Reverse transcriptase-polymerase chain reaction; Recombinant DNA

1. Introduction

The ability to detect and respond to chemical signals is a function possessed by the simplest forms of life. The family of molecular receptors for chemical ligands appear to have evolved from primordial receptor proteins. A large class of these molecular receptors share the c o m m o n characteristics of spanning the membrane seven times (7TM receptors) and being coupled to a G-proteinmediated signal transduction pathway (Strosberg, 1991). In sensory systems that respond to external stimuli, such

* Corresponding author. Present address: Department of Neurobiology, Bryan Research Building, Duke University Medical Center, Durham, NC 27710, USA. Tel.: (1-919) 681-5571; Fax: (1-919) 684-4431. This paper is dedicated to the memory of Dr. Richard Akeson, who passed away on April 14, 1993. 2 Present address: The Procter and Gamble Company, Miami Valley Labs, Cincinnati, OH, USA. Abbreviations: bp, base pair; cDNA, DNA complementary to RNA; DNase, deoxyribonuclease;G-protein, GTP binding protein; nt, nudeotide(s}; oligo, oligodeoxyribonucleotide;RT-PCR, reversetranscriptase-polymerase chain reaction; RNase, ribonuclease; SSC, 0.15M NaC1/0.015M Na3.citrate pH7.6; 7 TM, seven transmembrane domain; TB, taste bud.

as vision, olfaction and taste, considerable advances have been made toward understanding the molecular processes of sensory function. Many of the putative receptors in these systems are believed to be members of this large family of G-protein-coupled 7TM receptors (Shepherd, 1991). In higher organisms, specialized cells express individual members of this 7TM receptor family: rhodopsin in the photoreceptor cells, odorant receptors in the olfactory neurons and taste receptors in taste bud cells. While the genes for the visual system's opsin proteins were the first to be cloned, many new members have been added to the G-protein-coupled, 7TM receptor family in recent years (Khorana, 1992; Hargrave and McDowell, 1992). A very large subset of this family was found to be expressed in the olfactory epithelium and are thought to play an important role in olfaction (Buck and Axel, 1991; Nef et al., 1992; Ngai et al., 1993a,b; Raming et al., 1993; Ressler et al., 1993; Selbie et al., 1992). A few genes have also been cloned from the taste system and are very similar to the olfactory receptors(Matsuoka et al., 1993; Abe et al., 1993a,b). There have also been reports of olfactory receptor-like sequences being expressed in sperm (Parmentier et al., 1992; Vanderhaeghen et al., 1993). The function of these proteins in this tissue remains unclear.

0378-1119/96/$15.00 Copyright © 1996 Published by Elsevier Science B.V. All rights reserved PH $0378-1119(96)00311-3

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~B. Thomaset al./Gene 178 (1996) 1-5

We report the cloning of three new chemosensory receptors initially detected in rat vallate taste bud mRNA. Reverse transcriptase PCR (RT-PCR) analyses demonstrated that the mRNAs for these three receptors are also expressed in olfactory and male reproductive tissue. These studies are the first unambiguous demonstration that identical 7TM receptor genes are expressed in multiple tissues.

2. Experimental and discussion 2.1. Identification of gustatory 7 T M sequences

We set out to determine if vallate taste tissue expressed olfactory receptor-like genes. Degenerate oligonucleotide primers were designed from conserved amino acid residues in and near the third and seventh transmembrane domains of the olfactory receptor gene family. They were used in PCR reactions containing cDNAs prepared from rat vallate taste bud mRNA. To control for the amplification of genes expressed in tissue contaminating the vallate taste bud preparation, cDNAs prepared from non-taste tongue epithelium and tongue muscle were used in separate PCR reactions. The heterogeneous pool of sequences was cloned into a PCR cloning vector and individual clones were subjected to dideoxythymidine sequencing through their coding strands. Clones unique to the vallate taste bud preparations were completely sequenced. Three of the clones examined, TB334, TB567 and TB641, were found to contain open reading frames of approximately 525 nucleotides (nt) whose deduced amino acid sequences shared 30-75% homology with previously identified olfactory receptor family members. The most direct method for determining the fulllength coding sequence of these three putative receptors was to utilize genomic libraries since all of the previously identified members of the olfactory receptor family have been shown to be intronless. Sequence analysis of the resulting genomic clones confirmed that the genes were intronless within the coding regions and that the deduced amino acid sequences aligned with other members of the 7 T M gene family cloned from taste, testis and olfactory tissues (Fig. 1). Searches of the GenBank nucleotide and protein databases revealed that the putative receptors encoded by the TB334, TB567 and TB641 genes are most similar to the previously identified members of the olfactory receptor gene family isolated from rat(Buck and Axel, 1991; Raming et al., 1993), mouse (Nef et al., 1992; Ressler et al., 1993), dog (Parmentier et al., 1992) and human (Selbie et al., 1992). 2.2. Expression in other tissues

To determine the expression pattern of the three receptor genes in various rat tissues, we chose the

method of RT-PCR. This method is sensitive enough to detect low levels of mRNA in small heterogeneous tissue samples such as vallate tissue. The products that are generated in the PCR reactions can be sequenced to confirm that they are the result of the specific amplification of the desired gene product. Specific primers for amino acids within transmembrane domain IV and for amino acids within the fourth extracellular and/or seventh transmembrane domain were designed for all three of the putative taste receptor genes (see Fig. 1). RT-PCR reactions were performed with these specific primers using mRNAs prepared from a wide variety of adult rat tissues. To confirm that PCR products obtained were the result of mRNA conversion to cDNA and not from genomic DNA contamination, samples from each DNase-digested RNA were also used directly in PCR without prior first strand synthesis. Southern blot analyses with the appropriate 32P-labeled probe revealed the expression of TB334, TB567 and TB641 in vallate taste tissue, olfactory epithelium, olfactory bulb and male reproductive tissues but in none of the other tissues analyzed (Fig. 2). Primers were also designed from two of the rat olfactory receptor genes, ORF12 and ORF5 (Buck and Axel, 1991), and were used in identical PCR experiments. Primers for ORF12 receptor (Fig. 2) and ORF5 receptor(data not shown) gave similar results. Although originally isolated from the rat olfactory epithelium, these receptors showed expression in both olfactory and male reproductive tissue. They were not detected in vallate taste tissue or in any other tissue analyzed. Control reactions using primers specific for the rat y-actin and 13-2 adrenergic receptor confirmed the integrity of all cDNAs used in these experiments (Fig. 2).

2.3. Sequence identity of the R T - P C R products

To rigorously test whether the pattern of expression shown in the RT-PCR experiments was the result of specific amplification of single gene products, the RT-PCR reaction products from taste, olfactory and testis cDNAs (Fig. 3), as well as genomic DNA (data not shown) were gel purified and both strands were sequenced using the appropriate 32p-end labeled PCR primers in Taq cycle sequencing. The sequences of the PCR products for each gene from all sources were identical, confirming that each set of PCR primers amplified the desired gene product. While the number of studies that have been published on this family of olfactory-like receptor genes continues to grow, the true functionality of this family remains to be discovered. Experiments demonstrating the expression of a large number of these receptor genes in the olfactory epithelium supports the speculation that they play a very important role in olfaction (Nef et al., 1992;

M.B. Thomas et al./Gene 178 (1996) 1-5

E1 I II II MRRNRNTSLDTVVTDFLLLGLAHPPNLRTFLFLVFLLIYILTQLGNLLILLTVWADPKLHAR~MYILLGVLSFLDMWLSSvIVP TB641 MENQSSVSEFFLRGISGFPEQQQLLYGLFLCMYLVTLTG~LIILAIGSDPHLH-TPMYFFLANLSFADHGLISSTVT TB334 MTQRNATEVTDFYLLGFGVQQNTQcvLFIvFF•IYVTSMvGNTG•ILLINTNSRLQ-TPMYFFLQNLAFvDIcYTSAITP TB567 MILNCNPFSGLFLSMYLVTVLGNLLIILAVSSNSHLH-NLMYFFLSNLSFVDICFISTTIP GUST27 MTEKNQTVVSEFVLLGLPIDPDQRDLFYALFLAMYvTTILGNLLIIVLIQLDSHLH-TPMYLFLSNLSFSDLCFSSVTMP DTMT MESGNSTRRFSSFFLLGFTENPQLHFLIFA~FLSMYLvTVLGNLLII~IITQSHLH-TPMYFFLANLSFVDICFTSTTIP ORFI2 MSSTNQSSVTEFLLLGLSRQPQQQQLLFLLFLIMYLATvLGNLLIILAIGTDSRLH-TPMYFFLSNLSFvDvcFSSTTVP ORF5

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TB641 TB334 TB567 GUST27 DTMT ORFI2 ORF5

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TB641 TB334 TB567 GUST27 DTMT ORFI2 ORF5

E3 V I3 Vl FRLPYcGPKEvDYFFCDIPAVLRLAcADTAINELVTFVDIGvVAAScFLLILLSYANIVHAILKIRTADGRRRAFSTCGSHLTv ARLS•CVVGEIAHFFCDvTSv•KLSCSDTHVNELvLSGFGGTVLMVPFVSIVISYVHIVFAvL•IQSSGGSSKAFSTCSSHLCV FSLSYCNSKNINHFFCDwPIISLSCSNTjINIML•IvFVGFNLTFTVLVIIFSYIYIMAAILKMSSTAGRKKTFSTCAS•LTA NELNFSRGTEIPHFFCELAQVLKVANSDTHINNVF•YvVTSLLGLIPMTGILMSYSQIASSLLK•SSSVSKYKAFSTCGSHLCV ARLCFCA-NTIPHFFCD•SALLKLACSDTQVNELvIFIMGGLILvIPFLLIITSYARIVSSILKvPSAIGICKvFSTCGSHLSV LQLTFCGDvKIPHFFCELNQLSQLTC•DNFPSHLIMNLVPVMLAAISFSGILYSYFKIVSSIHSISTVQGKYKAFSTCASHLSI ARLSFCADN•IPHFFCDGTPLLKLSCSDTHLNELMILTEGAwMVTPFVCILISYIHITCAvLRvSSPRGGWKSFSTcGSHLAV

252 245 247 228 246 248 247

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VI E4 Vll 14 VTVYYVPCIFIYLRAGS-KSS-FDGAVAVFYTVVTPLLNPLIYTLRNQEVNSALKRLRAGRGNVGGDK VCVFYGTLFSVYLFPSSVETTEKDVAAAAHYTWTPMLNPFIYSLRNKDIKGALKRLLSHRRILSS VTIFYGTLSYMYLQPHSDNSEENMKVASVFYGIVIPMLNPLIYSLRNKEVKEGFKAMSRRFLRMKSNP VSLFYGSATIvYFcSSvLHSTHKKMIASLMYTvISPM~NPFIYSLRNKDVKGALGKLFIRvASCPLWSKDFRPKFILKPERQSL VSLFYGTVIGLYLCPSANNSTVKETIMAMMYTVVTPMLNPFIYSLRNKDMKGALRRVlCRKKITFSV VSLFYSTGLGVYVSSAVVQSSHSAASASVMYTWTPMLNPFIYSLRNKDVKRALERLLEGNCKVHHWTG VCLFYGTVIAVYFNPSSSHLAGRDMAAAVMYAVVTPMLNPFIYSLRNSDMKAALRKVLAMRFPSKQ

318 311 315 312 313 317 313

Fig. 1. Alignment of TB334, TB567 and TB641 with representative olfactory receptor-like proteins. Amino-acid sequences of the three genes isolated from taste bud are aligned with a rat gustatory receptor (GUST27; Abe et al., 1993b), a receptor isolated from canine testis (DTMT; Parmentier et al., 1992) and two rat olfactory receptors (ORF12 and ORF5; Buck and Axel, 1991). Overlines are predicted transmembrane regions. Extracellular (El-4) and intracellular (I1-4) regions are indicated above the sequences. Asterisks indicate amino-acid identity in all seven proteins. Underlined amino acids indicate sequence used to generate specific oligos for PCR. Methods: Tissues were taken from 150-200 gram Sprague-Dawley rats. Methods for mRNA and cDNA preparation were essentially as previously described (Small and Akeson, 1990; Reyes et al., 1991) although some mRNAs were prepared as described in Chomczynski and Sacchi (1987). Total cellular RNA (10 gg) from each tissue was initially digested with RNase-free DNase (Boehringer-Mannheim or US Biochemical) for 60 min at 37°C in 15 gl of buffer containing 2 mM MgCI2. After heat inactivation at 95°C for 10 min and rapid cooling on ice, one-third of the RNAs were used in a cDNA reaction using AMV-reverse transcriptase (Life Sciences). Control reactions without RT were also performed t test for genomic DNA contamination, cDNAs were primed with oligo(dT) (Pharmacia) and two oligos that represent highly conserved residues in the carboxy portion of many 7TM proteins. The fully degenerate oligos are: D-(V,I,M)K-(S,R,Y,H,Q,N,K,D,E,C,G)-A and N-P-F-I-Y. cDNAs were then used in PCR with fully degenerate oligos designed to amplify 7TM sequences. Oligos coding for the amino-acid sequences M-A-Y-D-(R,S)-Y-V and M-(F,L)-N-P-F-I-Y were used in PCR reactions which consisted of 50 cycles of 94°C, 1 min; 52°C, 1 min; 72°C, 2 min. Gel-purified products of 500-700 base pairs (bp) were subjected to a second round of PCR with these same oligos. PCR products were cloned into the pT7Blue vector (Novagen). Sequencing was performed with the Taq cycle sequencing kit (BRL). Clones TB334, TB567 and TB641 were initially found only in vallate taste bud samples as compared to other non-sensory control tissues. Isolation of these genes was performed by standard screening methods using a rat genomic cosmid library (Chen et al., 1990) and a rat genomic lambda library (Stratagene). GenBank accession Nos. for sequences are U50947, U50948 and U50949.

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M.B. Thomas et al./Gene 178 (1996) 1-5

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Fig. 2. RT-PCR analysis of TB334. TB567 and TB641 expression in rat tissues. Representative RT-PCR reactions were analyzed by Southern blots to demonstrate the restricted expression pattern of TB334, TB567 and TB641 Other specific RT-PCR amplifications shown are an olfactory receptor IORF12: Buck and Axel. 19911 and two ubiquitously expressed genes. 13-2 adrenerglc receptor and y-cytoplasmic actin (Gocayne et al.. 1987: Brown et al.. 1990J. The RT samples (lanes 13 17) represent a test for genomic DNA contamination as the starting mRNAs did not receive AMV-RT in the cDNA reaction. All lanes with visible products were diluted ten-fold prior to loading. Methods: Tissues were taken from 150 200 g Sprague-Dawley rats. Protocol for mRNA preparation and cDNA synthesis are described in the legend to Fig. 1. Following cDNA synthesis, 0.5-1.0 ~tg of cDNAs were used in PCR with oligos that were specific for the individual genes. The sequence of the specific oligos for TB334, TB567 and TB641 are underlined in Fig. 1. The oligos for the y-cytoplasmic actin gene are from nt 965-0093 and nt 1488-1507 of the published sequence (Brown et al., 1990). The oligos for [3-2 adrenergic receptor were designed from nt 19-27 and nt 190 198 of the published sequence (Gocayne et al., 1987). The PCR program followed was: 94°C, 1 min; 62°C, 1 min; 72°C, 2 min; for 40 cycles. PCR products were electrophoresed on 1% agarose gels, denatured and transferred to GeneScreen Plus membranes (DuPont) according to manufacturer's protocol. Hybridization with specific 3Zp-labeled probes was carried out at 61°C overnight and washed at increasing stringencies. Final wash used was 0.5 × SSC at 61°C for 30 min. Hybridized membranes were exposed to X-OMAT AR film and developed with an X-OMAT M20 processor (Kodak).

R a m i n g et al., 1993; R e s s l e r et al., 1993, 1994; V a s s a r et al., 1993, 1994). T h e r o l e of r e c e p t o r s e x p r e s s e d in o t h e r tissues, s u c h as t h e testes, r e m a i n s u n k n o w n . A role for the r e c e p t o r s d e s c r i b e d in this r e p o r t c a n be e n v i s i o n e d in the sense o f taste, a l t h o u g h this i d e a w o u l d o n l y t r u l y be s u p p o r t e d by the d i s c o v e r y of t h e i r ligands.

TB641

Fig. 3. Sequence analysis of TB334, TB567 and TB641 PCR products from taste bud, olfactory epithelium and testis. RT-PCR products of all three genes were gel-purified and used directly in double-stranded sequencing reactions using the appropriate 32p-end-labeled PCR primers. Sequences for all three products were obtained for both the coding and non-coding strands. Shown are representative regions of the coding (TB334 and TB567) and non-coding (TB641) strands. All lanes are loaded G,A,T,C. Methods: Preparation of mRNAs and cDNAs are described in the legend to Fig. i. PCR was performed using 0.5 1.0 Ixg of the cDNAs as templates and oligos specific for the TB genes underlined in Fig. 1. PCR products were gel-purified and used directly in double-stranded sequencing reactions using the appropriate 32p-end-labeled oligo. Sequencing reactions were electrophoresed on denaturing 6% acrylamide gels and autoradiographed overnight on Hyperfilm MP (IBI). Sequences were analyzed using both Mac Vector (IBI) and Microgenie (Beckman) sequence analysis software.

3. Conclusions (1) W e h a v e b e e n a b l e to c l o n e t h r e e n e w m e m b e r s of the large 7TM, G-protein-coupled receptor family u s i n g a s t r a t e g y t h a t c o m b i n e s the use of d e g e n e r a t e oligonucleotides with RT-PCR. These new members, T B 3 3 4 , T B 5 6 7 a n d T B 6 4 1 , w e r e i s o l a t e d f r o m rat v a l l a t e taste b u d m R N A a n d are m o s t s i m i l a r to members of the olfactory receptor subfamily. (2) W h i l e the n e w g e n e s w e r e i s o l a t e d b y v i r t u e of their e x p r e s s i o n in rat v a l l a t e taste tissue, R T - P C R a n a l y -

M.B. Thomas et al./Gene 178 (1996) 1-5

sis shows expression of these genes not only in the taste bud but also in olfactory epithelium and in the male reproductive tract. (3) RT-PCR with specific primer sets followed by sequencing of the amplified products demonstrated that the PCR products generated in the reaction were the result of amplification of a single gene product. This experiment demonstrates that transcripts from any one of the three genes can be detected in each of these tissues.

Acknowledgement We are grateful to Shirley Arnold and Fred Rosenberry for technical assistance in the early phases of this project. We thank Drs. Brian Key, Dan Wiginton, Sandra Degen and James Lessard for their helpful advice. We also thank Dennis Thomas for critical review of the manuscript. This work was supported by the NIH grant DC00347.

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Hargrave, P.A. and McDowell, J.H. (1992) Rhodopsin and phototransduction: a model system for G protein-linked receptors. FASEB J. 6, 2323-2331. Khorana, H.G. (1992) Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J. Biol. Chem. 267, 1-4. Matsuoka, I., Mori, T., Aoki, J., Sato, T. and Kurihara, K. (1993) Identification of novel members of G-protein coupled receptor superfamily expressed in bovine taste tissue. Biochem. Biophys. Res. Commun. 194, 504-511. Nef, P., Hermans-Borgmeyer, I., Artieres-Pin, H., Beasley, L., Dionne, V.E. and Heinemann, S.F. (1992) Spatial pattern of receptor expression in the olfactory epithelium. Proc. Natl. Acad. Sci. USA, 89, 8948 8952. Ngai, J., Chess, A., Dowling, M.M., Necles, N., Macagno, E.R. and Axel, R. (1993a) Coding of olfactory information: topography of odorant receptor expression in the catfish olfactory epithelium. Cell 72, 667 680. Ngai, J., Dowling, M.M., Buck, L., Axel, R. and Chess, A. (1993b) The family of genes encoding odorant receptors in the channel catfish. Cell 72, 657 666. Parmentier, M., Libert, F., Schurmans, S., Schiffmann, S., Lefort, A., Eggerickx, D., Ledent, C., Mollereau, C., Gerard, C., Perret, J. et al. (1992) Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature 355, 453-435. Raming, K., Krieger, J., Strotmann, J., Boekhoff, I., Kubick, S., Baumstark, C. and Breer, H. (1993) Cloning and expression of odorant receptors. Nature 361,353 356. Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73, 597 609. Ressler, K.J., Sullivan, S.L. and Buck, L.B. (1994) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245--1255. Reyes, A.A., Small, S.J. and Akeson, R. (1991) At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are expressed during rat heart development. Mol. Cell. Biology 11, 1654 1661. Selbie, L.A., Townsend-Nicholson, A., Iismaa, T.P. and Shine, J. (1992) Novel G protein-coupled receptors: a gene family of putative human olfactory receptor sequences. Mol. Brain Res. 13, 159-163. Shepherd, G.M. (1991) Sensory transduction: entering the mainstream of membrane signaling. Cell 67, 845-851. Small, S.J. and Akeson, R. (1990) Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development. J. Cell Biol. 111, 2089-2096. Strosberg, A.D. (1991) Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins. Eur. J. Biochem. 196, 1 10. Vanderhaeghen, P., Schurmans, S., Vassart, G. and Parmentier, M. (1993) Olfactory receptors are displayed on dog mature sperm cells. J. Cell Biol. 123, 1441 1452. Vassar, R., Ngai, J. and Axel, R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74, 309 318. Vassar, R., Chao, S.K., Sitcheran, R., Nunez, J.M., Vosshall, L.B. and Axel, R. (1994) Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981 891.